Characterization of glucose-specific catabolite repression-resistant mutants of Bacillus subtilis: identification of a novel hexose:H+ symporter.
Insertional mutagenesis was conducted on Bacillus subtilis cells to screen for mutants resistant to catabolite repression. Three classes of mutants that were resistant to glucose-promoted but not mannitol-promoted catabolite repression were identified. Cloning and sequencing of the mutated genes revealed that the mutations occurred in the structural genes for (i) enzyme II of the phosphoenolpyruvate-glucose phosphotransferase (PtsG), (ii) antiterminator GlcT, which controls PtsG synthesis, and (iii) a previously uncharacterized carrier of the major facilitator superfamily, which we have designated GlcP. The last protein exhibits greatest sequence similarity to the fucose:H+ symporter of Escherichia coli and the glucose/galactose:H+ symporter of Brucella abortus. In a wild-type B. subtilis genetic background, the glcP::Tn10 mutation (i) partially but specifically relieved glucose- and sucrose-promoted catabolite repression, (ii) reduced the growth rate in minimal glucose medium, and (iii) reduced rates of [14C]glucose and [14C]methyl alpha-glucoside uptake. In a delta pts genetic background no phenotype was observed, suggesting that expression of the glcP gene required a functional phosphotransferase system. When overproduced in a delta pts mutant of E. coli, GlcP could be shown to specifically transport glucose, mannose, 2-deoxyglucose and methyl alpha-glucoside with low micromolar affinities. Accumulation of the nonmetabolizable glucose analogs was demonstrated, and inhibitor studies suggested a dependency on the proton motive force. We conclude that B. subtilis possesses at least two distinct routes of glucose entry, both of which contribute to the phenomenon of catabolite repression.
Paulsen
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Department of Biology, University of California at San Diego, La Jolla 92093-0116, USA.
Chauvaux
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S
Choi
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Saier
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MH
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D001426:Bacterial Proteins; D001483:Base Sequence; D001692:Biological Transport; D002250:Carbon Radioisotopes; D002352:Carrier Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; D003847:Deoxyglucose / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D008757:Methylglucosides / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D016254:Mutagenesis, Insertional; D009876:Operon; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D027981:Symporters; D015534:Trans-Activators / Q000235:genetics
D001426:Bacterial Proteins; D002250:Carbon Radioisotopes; D002352:Carrier Proteins; D004251:DNA Transposable Elements; D004269:DNA, Bacterial; C095882:GltC protein, Bacillus subtilis; D005942:Gluconates; D008565:Membrane Proteins; D008757:Methylglucosides; D009004:Monosaccharide Transport Proteins; D012097:Repressor Proteins; D027981:Symporters; D015534:Trans-Activators; C089820:monosaccharide-hydrogen cotransporter; C027020:methylglucoside; D003847:Deoxyglucose; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C031629:phosphoenolpyruvate-glucose phosphotransferase; D005947:Glucose; C030691:gluconic acid